Abstract
Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4–7 min window. This polarized mRNA trafficking depended on the 3′ untranslated region (UTR) since mRNA-Δ3′UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated “worm-like” trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3′UTR is essential for this cue-induced regulation.
Highlights
IntroductionLocal protein synthesis can be elicited rapidly by extrinsic cues and is required in axons for a wide range of specialized activities such as directional turning, branching, maintenance, and repair (Campbell and Holt, 2001; Verma et al, 2005; Wu et al, 2005; Leung et al, 2006; Piper et al, 2006; Yao et al, 2006; Cox et al, 2008; Spillane et al, 2012; Yoon et al, 2012; Cosker et al, 2013; Donnelly et al, 2013; Wong et al, 2017; Cagnetta et al, 2018; Terenzio et al, 2018)
To investigate how extracellular stimuli affect the trafficking of identified mRNAs in growing axons, we used the approach of introducing fluorescent-labeled mRNAs into retinal ganglion cell (RGC) neurons
We found that cyanine 3 (Cy3)-labeled mRNA encoding green fluorescent protein (GFP) gave rise to a GFP signal in embryos and in cultured RGC growth cones (Figure S1), indicating that synthetic mRNA can be recognized by the translation machinery and translated into protein
Summary
Local protein synthesis can be elicited rapidly by extrinsic cues and is required in axons for a wide range of specialized activities such as directional turning, branching, maintenance, and repair (Campbell and Holt, 2001; Verma et al, 2005; Wu et al, 2005; Leung et al, 2006; Piper et al, 2006; Yao et al, 2006; Cox et al, 2008; Spillane et al, 2012; Yoon et al, 2012; Cosker et al, 2013; Donnelly et al, 2013; Wong et al, 2017; Cagnetta et al, 2018; Terenzio et al, 2018). Growth cone turning toward an attractive gradient of Netrin-1, brain derived neurotrophic factor (BDNF) or Sonic Hedgehog (SHH) requires the asymmetric synthesis of β-actin protein on the near-side of the growth cone, closest to the source (Leung et al, 2006; Yao et al, 2006; Lepelletier et al, 2017), facilitating cytoskeletal assembly in the direction of growth The mechanism underlying this spatially polarized protein synthesis is poorly understood but likely involves mRNA localization and localized translation since this highly conserved mechanism occurs across species and cell types to ensure positioning of nascent proteins precisely where they are needed. These localization studies were done at single time points in fixed samples and it is not clear whether the ribonucleoprotein particle (RNP) behavior brings about the changes in mRNA distribution (Bullock, 2011; Doyle and Kiebler, 2011) or whether there is cue-polarized transport
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